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A light-addressable potentiometric sensor (LAPS) is a field-effect-based potentiometric sensor with an electrolyte/insulator/semiconductor (EIS) structure, which is able to monitor analyte concentrations of (bio-)chemical species in aqueous solutions in a spatially resolved way. Therefore, it is also an appropriate tool to record 2D-chemical images of concentration variations on the sensor surface. In the present work, two differential, LAPS-based measurement principles are introduced to determine the metabolic activity of Escherichia coli (E. coli) K12 and Chinese hamster ovary (CHO) cells as test microorganisms. Hereby, we focus on i) the determination of the extracellular acidification rate (ΔpH/min) after adding glucose solutions to the cell suspensions; and ii) recording the amplitude increase of the photocurrent (Iph) related to the produced acids from E. coli K12 bacteria and CHO cells on the sensor surface by 2D-chemical imaging. For this purpose, 3D-printed multi-chamber structures were developed and mounted on the planar sensor-chip surface to define four independent compartments, enabling differential measurements with varying cell concentrations. The differential concept allows eliminating unwanted drift effects and, with the four-chamber structures, measurements on the different cell concentrations were performed simultaneously, thus reducing also the overall measuring time.
The incorporation of nanomaterials that are biocompatible with different types of biological compounds has allowed the development of a new generation of biosensors applied especially in the biomedical field. In particular, the integration of film-based nanomaterials employed in field-effect devices can be interesting to develop biosensors with enhanced properties. In this paper, we studied the fabrication of sensitive nanofilms combining ZnO nanocrystals and carbon nanotubes (CNTs), prepared by means of the layer-by-layer (LbL) technique, in a capacitive electrolyte-insulator-semiconductor (EIS) structure for detecting glucose and urea. The ZnO nanocrystals were incorporated in a polymeric matrix of poly(allylamine) hydrochloride (PAH), and arranged with multi-walled CNTs in a LbL PAH-ZnO/CNTs film architecture onto EIS chips. The electrochemical characterizations were performed by capacitance–voltage and constant capacitance measurements, while the morphology of the films was characterized by atomic force microscopy. The enzymes glucose oxidase and urease were immobilized on film’s surface for detection of glucose and urea, respectively. In order to obtain glucose and urea biosensors with optimized amount of sensitive films, we investigated the ideal number of bilayers for each detection system. The glucose biosensor showed better sensitivity and output signal for an LbL PAH-ZnO/CNTs nanofilm with 10 bilayers. On the other hand, the urea biosensor presented enhanced properties even for the first bilayer, exhibiting high sensitivity and output signal. The presence of the LbL PAH-ZnO/CNTs films led to biosensors with better sensitivity and enhanced response signal, demonstrating that the adequate use of nanostructured films is feasible for proof-of-concept biosensors with improved properties that may be employed for biomedical applications.
The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33–43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.
Light-addressable potentiometric sensors for quantitative spatial imaging of chemical species
(2017)
A light-addressable potentiometric sensor (LAPS) is a semiconductor-based chemical sensor, in which a measurement site on the sensing surface is defined by illumination. This light addressability can be applied to visualize the spatial distribution of pH or the concentration of a specific chemical species, with potential applications in the fields of chemistry, materials science, biology, and medicine. In this review, the features of this chemical imaging sensor technology are compared with those of other technologies. Instrumentation, principles of operation, and various measurement modes of chemical imaging sensor systems are described. The review discusses and summarizes state-of-the-art technologies, especially with regard to the spatial resolution and measurement speed; for example, a high spatial resolution in a submicron range and a readout speed in the range of several tens of thousands of pixels per second have been achieved with the LAPS. The possibility of combining this technology with microfluidic devices and other potential future developments are discussed.
The capacitive electrolyte–insulator–semiconductor (EIS) structure is a typical device based on a field-effect sensor platform. With a simple silicon-based structure, EIS have been useful for several sensing applications, especially with incorporation of nanostructured films to modulate the ionic transport and the flat-band potential. In this paper, we report on ion transport and changes in flat-band potential in EIS sensors made with layer-by-layer films containing poly(amidoamine) (PAMAM) dendrimer and single-walled carbon nanotubes (SWNTs) adsorbed on p-Si/SiO 2 /Ta 2 O 5 chips with an Al ohmic contact. The impedance spectra were fitted using an equivalent circuit model, from which we could determine parameters such as the double-layer capacitance. This capacitance decreased with the number of bilayers owing to space charge accumulated at the electrolyte–insulator interface, up to three PAMAM/SWNTs bilayers, after which it stabilized. The charge-transfer resistance was also minimum for three bilayers, thus indicating that this is the ideal architecture for an optimized EIS performance. The understanding of the influence of nanostructures and the fine control of operation parameters pave the way for optimizing the design and performance of new EIS sensors.